1. Field of the Invention
The invention is generally related to the use of aryl compounds with one or more aldehydic moieties and one or more carboxylic acid moieties to allosterically modify hemoglobin. More particularly, the invention is directed to a method of allosterically modifying hemoglobin with aryl compounds having multiple functional groups, such as aldehydes and carboxylic acids, to stereospecifically cross-link .alpha. subunits of the hemoglobin molecule.
2. Description of the Prior Art
Hemoglobin is a tetrameric protein which delivers oxygen via an allosteric mechanism. Oxygen binds to the four hemes of the hemoglobin molecule. Each heme contains porphyrin and iron in the ferrous state. The ferrous ion-oxygen bond is readily reversible. Binding of the first oxygen to a heme requires much greater energy than the second oxygen molecule, binding the third oxygen molecule requires even less energy, and the fourth oxygen requires the lowest energy for binding. Hemoglobin has two .alpha. and two .beta. subunits arranged in two fold symmetry. The .alpha. and .beta. dimers rotate during oxygen release to open a large central water cavity. It is generally understood that the allosteric transition that involves the movement of the alpha-beta dimer takes place between the binding of the third and fourth oxygen, and the .alpha..sub.1 .beta..sub.1 interface binding is tighter than the .alpha..sub.1 .alpha..sub.2 or .alpha..sub.1 .beta..sub.2 interfaces.
The chemical and physical structure of hemoglobin has been well characterized in the art. Several references, including (Bunn et al., Human Hemoglobins, W. B. Saunders Company, London, 1977, and Fermi et al., Atlas of Molecular Structures in Biology, ed. Phillips, D.C., Vol. 2, Clarendon Press, Oxford, 1981, which are both herein incorporated by reference) set forth the amino acid sequences of the hemoglobin molecule.
In blood, hemoglobin is in equilibrium between two allosteric structures. In the "T" (for tense) state, hemoglobin is de-oxygenated. In the "R" (for relaxed) state, hemoglobin is oxygenated. An oxygen equilibrium curve can be scanned using well known equipment such as the the Aminco.TM. Hem-o-scan to evaluate the affinity and degree of cooperativity (allosteric action) of hemoglobin. In the scan, the Y-axis plots the percent of hemoglobin oxygenation and the X-axis plots the partial pressure of oxygen in millimeters of mercury (mm Hg). If a horizontal line is drawn from the 50% oxygen saturation point to the scanned curve and a vertical line is drawn from the intersection point of the horizontal line with the curve to the partial pressure X-axis, a value commonly known as the P.sub.50 is determined. This is the pressure when the hemoglobin is 50% saturated with oxygen. By comparing the P.sub.50 value for treated hemoglobin to the P.sub.50 value for untreated normal adult human hemoglobin (HbA) under physiological conditions (i.e., 37.degree. C., pH=7.4, and partial carbon dioxide pressure of 40 mm Hg), the degree of allosteric modification can be determined. When the P.sub.50 value for treated hemoglobin is lower than for HbA, a high oxygen affinity hemoglobin is indicated and the curve is said to be "left-shifted". Conversely, when the P.sub.50 value for treated hemoglobin is higher than for HbA, a low oxygen affinity hemoglobin is indicated and the curve is said to be "right-shifted".
Influencing the allosteric equilibrium of hemoglobin is a viable avenue of attack for treating a wide variety of diseases. For example, several new compounds and methods for treating sickle cell anemia which involve the allosteric regulation of hemoglobin are reported in U.S. Pat. Nos. 4,699,926 to Abraham et al., 4,731,381 to Abraham et al., 4,731,473 to Abraham et al., 4,751,244 to Abraham et al., 4,887,995 to Abraham et al. In addition, new classes of allosteric hemoglobin modifiers described in U.S. Pat. Nos. 5,290,803 to Abraham et al., 5,122,539 to Abraham et al., 5,049,695 to Abraham et al., and 5,248,785 to Abraham et al., have been shown to restore the oxygen carrying capacity of stored blood, and to be useful in treating a variety of disorders such as ischemia, radiotherapy of tumors, respiratory distress syndromes, wound healing, etc.
Aldehydes such as vanillin and 5-(2-formyl-3-hydroxyphenoxy)pentanoic acid have been shown to react with hemoglobin and possess antisickling activity (See, Kreen, G., White, R. D., Br. J. Pharmacol., 1981, 74, 965, and Zaugg, R. H.; Walter, J. A.; Klotz, I. M., J. Biol. Chem., 1977, 252, 8542).